PROJECT SUMMARY Determining the molecular mechanisms mediating the health benefits of exercise is a challenging, but extremely important undertaking, that can lead to the identification of novel therapeutic targets. The significance of this area of research is underscored by the large-scale NIH initiative ?Molecular Transducers of Physical Activity Consortium? (MoTrPAC), which aims to understand the molecular footprint of exercise in healthy humans and rats. While the extensive, in-depth data provided by MoTrPAC will be a landmark project, this consortium will only study healthy humans and rodents. Thus, the molecular footprint of exercise under conditions of obesity and other metabolic diseases may remain largely unknown, even in the post-MoTrPAC era. The overall goal of this project is to use state-of-the art omics platforms to discover the mechanism by which exercise training improves health under conditions of metabolic disease. Specific Aims are to determine: a) tissue-specific changes in cell types and cell composition in response to exercise and diet; b) tissue-specific transcriptional responses and activation of molecular pathways that can reverse the unfavorable metabolic effects of diet- induced obesity; and c) cell-type and tissue crosstalk in both lean and obese mice. Analysis of these complex data using computational biology tools has the power to unravel the molecular basis of disease and identify therapeutic targets. This large-scale project has two phases. The first phase includes the collection of multiple tissues from insulin resistant mice treated with or without exercise and second phase is multi-omics and bioinformatics analysis of these samples. The first phase of this project has recently been completed by the applicant, allowing for a feasible 2-year research plan to complete the second phase of the project. For the first phase, mice were divided in four groups: sedentary chow-fed; trained chow-fed; sedentary high fat diet-fed; and trained high fat diet-fed. Diet treatments were for 6 weeks and exercise training was done by housing mice with free access to a running wheel for 3 weeks. Seven tissues known to play significant roles in metabolism were collected: triceps, subcutaneous and visceral white adipose tissue, small intestine, hypothalamus, hippocampus and brain cortex. Phase 2 of this project is to perform single-cell transcriptomics and metabolomics on all collected tissues followed by data analysis and integration using computational biology tools. The applicant?s training plan will include dedicated mentorship by both Dr. Laurie Goodyear (sponsor) in the areas of exercise, metabolism, and diabetes, and Dr. Manolis Kellis (co-sponsor) for multi-omic approaches and computational biology. The applicant will also complete formal bioinformatics training through Harvard and MIT. These activities will provide her with the necessary tools critical for development as an independent physician scientist. This project will not only provide an outstanding training opportunity for the applicant, but should also lead to discoveries with broad implications for the treatment of obesity and diabetes.